PDGF is a mitogen which is released from the alpha-granules of platelets following activation. The mitogenic activity of this growth factor is restricted to cells of mesothelial origin, glial cells and fibroblasts, but excluding arterial endothelium cells.
PDGF isolated from blood is predominantly a disulphide-linked heterodimer having a molecular weight of 28000-31000 and is composed of two chains, the A chain (PDGF-A) and the B chain (PDGF-B). The mature A and B chains show 60% homology and the 8 cysteine residues in each chain are conserved. The PDGF B chain (SEQ ID NO: 1) is essentially homologous to the v-sis oncogene product, p28.sup.sis, derived from simian sarcoma virus. The major form of PDGF found in human serum is the heterodimer of the A and B chains, but homodimers are also present in small quantities. Homodimers of PDGF-BB are found in porcine.serum and PDGF-AA is produced by several human tumour cell Lines. The reduced monomeric forms of PDGF are biologically inactive.
PDGF receptors are composed of two sub-units, alpha and beta. The beta-receptor sub-unit can only bind the B chain of PDGF, but the alpha-receptor sub-unit binds both the A and the B chains and therefore it is possible to mediate the different biological functions of PDGF via the isoforms and the porportion of alpha-and beta-receptor sub-units on cells.
There is no structural model of PDGF and the residues responsible for binding to the receptor have not been defined. Circular dichroism measurements have shown a high content of random structure and a low alpha helical content (Vogel and Hoppe 1989, Biochemistry 28, 2961-2966). The minimal v-sis transforming domain has been shown to span the 89 residues indentical to PDGF and containing all 8 conserved cysteines (King et al 1985, P.N.A.S., 82, 5259-5299). Chimeras of PDGF-A and B chains have been used to define a region from residues 24-63 (numbering from the mature N-terminus) to be responsible for the transforming ability of PDGF-BB (LaRocheHe et al, 1990, Science, 248, 1541-1544). More recently, using single amino acid deletions, the receptor binding domain has been shown to be between residues 25 and 37 from the mature N-terminus. (Giese et al, 1990, M.C.B., 10, 5496-5501).
PDGF has been implicated in a number of diseases involving abnormal cell proliferation, inflammation, fibrosis, atherosclerosis and neoplasia. In addition, PDGF is thought to be an important component of the wound healing process. There is therefore considerable interest in the therapeutic potential of PDGF antagonists and agonists. The production of large quantities of PDGF is therefore desirable and the simplest way to achieve this is by recombinant DNA technology.
Recombinant PDGF is known and has been described in several prior publications for example U.S. Pat. No. 4769328 and U.S. Pat. No. 4801542, describe biologically active PDGF analogues expressed in yeast.
U.S. Pat. No. 4766073 discloses dimetic proteins substantially homologous to the A-chain or the B-chain of PDGF or portions thereof, and an A-B heterodimer, expressed in yeast. In addition a portion of the DNA sequence may encode a portion of the A-chain while another may encode a portion of the B-chain.
U.S. Pat. No. 4845075 discloses dimeric proteins which have substantially the same biological activity as PDGF. The proteins consist of dimers of polypeptides which are substantially homologous to PDGF-B and the specification suggests that it may be advantageous to truncate the protein or to change the amino acid residues. In particular, Cys residues may be substituted by other amino acid residues.
More specifically a truncated form of PDGF is disclosed. The Lys-Arg at the alpha factor B-chain boundary was removed, and it was proposed that proteolytic processing would occur at the internal Arg-Arg site at position 27-28. Mitogenic activity was observed with this construct. No N-terminal sequence analysis or SDS-PAGE analysis was performed on this construct and therefore it is not known at what position the processing took place. It is probable that an alternative site within alpha factor or PDGF-B was used since a molecule not containing the receptor binding region would have very low mitogenic activity.
EP-A-0282317 describes two forms of the PDGF-A chain, and PDGF-A-chain/B-chain heterodimers. The polypeptides were expressed in yeast and produced active PDGF dimers.
However, there is a major problem associated with the production of recombinant DNA which has not been addressed in the prior art and this is that the expression of the PDGF B chain, particularly in yeast, is very low. Yeasts are particularly suitable organisms for the expression of PDGF since, unlike E. coli, they contain proteases which allow maturation cleavage of the polypeptide chains. In addition, unlike transformed mammalian cells, they do not contain oncogenes. It would therefore be advantageous to develop a strategy for increasing the yield of recombinant PDGF-B in yeast.
It has previously been shown that both PDGF-A and PDGF-B undergo proteolytic processing during expression and secretion in vivo and that the mature A chain has 104 amino acid residues whilst the mature B chain has 109 amino acid residues.
However, it has been observed that recombinant PDGF is cleaved by proteases not only at the normal matruation site but also at various other sites along the chain an it is probable that this additional cleavage is to a large extent responsible for the low expression of recombinant PDGF-B in yeast. If this cleavage could be eliminated, then it is likely that the expression of PDGF-B would be greatly
EP-A-0282317 relates to polypeptides expressed in CHO cells having a sufficient part of the structural conformation of PDGF to bind to monoclonal antibodies specific for epitopes of the B chain of PDGF. N-terminal amino acid sepuence analysis of the recombinant PDGF produced in CHO cells reveals a small percentage of a terminus begining at amino acid 33 Thr-Asn-Ala-Asn-Phe and at amino acid 80 Lys-Lys-Pro-Ile-Phe. Similar processing has been observed. in platelets. These products were reasoned to be due to the action of specific proteolytic enzymes found inside CHO cells and platelets. The authors suggested that the cleavage site residues and adjacent residues may be altered to prevent cleavage.
It has already been demonstrated that it is possible to disrupt the protease cleavage sites of polypeptides or proteins by the substitution of an amino acid residue at the cleavage site with another amino acid residue.
The amino acid at the cleavage site is usually replaced by an amino acid having a similar chemical nature. However, problems can arise with this approach since, on the one hand, it is important to ensure that the cleavage site is actually disrupted whilst on the other hand, the protein must retain its biological activity and it is often difficult to modify the protein in such a way that it meets both of